The goal of this project is to determine the sequence of cellular events in hepatocarcinogenesis. In situ hybridization of albumin c-DNA and RNA probes for the albumin and alpha-fetoprotein genes to the hepatocytes in preneoplastic foci and in hepatocellular carcinomas revealed a decreased expression. Albumin expression is also decreased in the centrilobular areas of liver after phenobarbital administration. alpha-Fetoprotein is not expressed in early preneoplastic lesions but occasionally it is expressed in groups of cells inside the neoplastic nodule. We were able to transplant hepatocytes obtained from the individual nodules by needle biopsies to the anterior chamber of the rat eye. In order to study events in chemical hepatocarcinogenesis in vitro, a tissue culture system, which preserves the differentiated state of hepatocyes, is a necessity. We have demonstrated maintenance of asialoglycoprotein receptor, which we regard as an excellent marker for fully differentiated hepatocyte, for an extended priod of time, when hepatocytes were cocultured with rat liver epithelial cell line. Treatment of rat liver epithelial cells with TGF-beta increased the number of binucleated cells, the expression of albumin and the size of these cells.